The fibrotic scar that results after a myocardial infarction (MI) is stiff extracellular matrix (ECM), owing to the enhanced secretion of collagen as the tissue thins and undergoes necrosis. Pervious methods to improve myocardial function post-MI, e.g. cardiac patches and cell injections, do not sufficiently mimic the intrinsic properties of the matrix, such as stiffness (denoted E). Moreover, they often employ adult stem cells which have not been shown to have significant remodeling capacity and instead are more responsive to aberrant matrix conditions;thus cells have been observed to improperly differentiate into osteoblast-like cells or to fail to differentiate altogether in infarcted myocardium that is 3-fold too stiff, i.e. EInfarct >>ECARDIO. We have recently developed a dynamic, thiol-modified hyaluronic acid (HA-S)-based hydrogel that displays developmentally appropriate stiffness over time via time-dependent crosslinking. We have also shown that this can improve cardiac progenitor differentiation in mature cardiomyocytes by nearly an order of magnitude over soft matrix that does not remodel. In this proposal, we will first extend our findings to embryonic stem cells (ESCs), which should be even more effective at matrix remodeling than previous stem cell types. Expression of cardiac-specific genes in 2D and 3D HA-S hydrogels will first be monitored in ESCs to determine if HA-S can induce cardiomyogenesis relative to cardiac progenitor cells and age-matched control animals, and if not, at least ensure that it enhances differentiation over HA-S hydrogels that have had their time-dependent crosslinking removed by treatment with iodacetamide. Matrix secretion, assembly, and remodeling (via degradation by hyaluronidase) will also be measured and compared to cardiac progenitor cells to ensure that ESCs can indeed remodel matrix effectively and that cells can migrate sufficiently in the material. Cells and HA-S hydrogels will subsequently be used in a subcutaneous rat model to ensure biocompatibility and monitor hydrogel properties in vivo, e.g. time-dependent stiffening. Finally in a rat model of MI, ESCs and/or cardiac progenitor will be used in conjunction with the HA-S to determine to what degree our HA-S hydrogel can improve myocardial function post-MI versus convention treatments, e.g. cell injection. PUBLIC HEALTH RELEVANCE: As a leading cause of death in the United States, congestive heart failure (CHF) post-myocardial infarction (MI) has incited the need to develop novel techniques that prevents the formation of a stiff, scarred muscle wall which impairs heart function. Over the past two decades, novel strategies using stem cell patches or injections have not been able to sufficiently remodel the tissue and restore its function, which may be in part due to their inability to address specific design criteria of the diseased niche, e.g. stiffness (denoted E);often the addition of adult stem cells into this niche results in stem cells responding to the environment rather than remodeling it, and as such, aberrant stem cell behavior occurs given that the niche is 3- to 4-fold too stiff, EInfarct, relative to healthy muscle, ECARDIO. Materials have also been proposed in conjunction with cells to treat MI, but they often do not adequately mimic native myocardial design requirements or use cells with sufficient capacity to remodel the niche. In response, we have engineering a hyaluronic acid (HA)-based material that has time-dependent crosslinking to specifically mimic how the myocardium stiffens from soft embryonic stem cells (ESCs), EESC, to the stiffer heart wall, ECARDIO;time-dependent stiffening induces cardiac progenitors to express 3-fold more mature cardiac markers versus static cultures. It also causes 85% of progenitors to form mature, contractile sarcomeres versus only 15% for static cultures. When combined with ESCs that are uniquely tuned to remodel and shape their niche as they develop, this HA- and ESC-based approach may be able to sufficiently protect ESCs as they develop, migrate into the scarred host myocardium, and subsequently remodel it to attempt to restore some level of contractile function. In this proposal, we will first study to what extent the engineered HA material enhances cardiac differentiation and matrix secretion, assembly, and remodeling in ESCs. After understanding this interaction and better, the combination will be introduced subcutaneously in a rat model to determine biocompatibility and then in used in a rat infarct model to assess its ability to improve cardiac outcome.